This invention relates compositions and methods for making swine embryonic stem cells, chimeric swine from the stem cells, and transgenic swine from the chimeras.
Although transgenic animals have been produced by several different methods in several different species, methods to readily and reproducibly produce transgenic large mammals at reasonable costs are still lacking.
Methods to produce transgenic swine, for example, by microinjecting swine ova with genetic material, is very expensive. It costs between $25,000 to $250,000 to produce a single transgenic line. Another problem is that microinjection is a technically difficult procedure with an unacceptably low success rate. Furthermore, DNA transferred by microinjection is incorporated at random in the genome, usually in tandem linear arrays of multiple copies of the transgene. These limitations have resulted in animals being produced where 1) the transgene is not incorporated at all, 2) the transgene is incorporated but not expressed, 3) the transgene is incorporated but expressed transiently or aberrantly, and rarely 4) the transgene is incorporated and expressed normally. Also the incorporation of transgenes in the host genome may result in the disruption of an endogenous gene by a so-called insertional mutation, where some aspect of its development, growth or normal physiology is disrupted. Furthermore, this random insertion results in difficulties in controlling how the transgene will be regulated because flanking sequences upstream and downstream of the inserted transgenic DNA construct and which provide the control are randomly associated with the transgene.
One of the methods to generate transgenic animals, the use of transformed embryonic stem cells (ES-cells), has shown certain advantages over other methods when used to produce mouse chimeras, from which transgenic mice are derived. Once isolated, ES-cells may be grown in vitro for many generations producing unlimited numbers of identical cells capable of developing into fully formed adult chimeras. (Bradley et al., 1984).
A second major advantage of ES-cells is that they can be genetically manipulated in vitro. ES-cells may be transformed by introducing exogenous DNA into the cells via electroporation. Following transformation individual ES-cell clones may be screened in vitro for the incorporation of the exogenous DNA before being used to produce chimeric embryos (Thomas et al., 1987). ES-cell clones containing the transferred DNA can be selected and used for blastocyst injection. The ability to screen and select transformed ES-cells in vitro is one of the most important features for utilizing this strategy to produce transgenic animals.
When transformed ES-cells are used to make chimeric embryos, some of these cells may be incorporated into the gonads and participate in the formation of sperm and ova. Incorporation of the transgene into the gametes permits germ line transmission. Some of the offspring produced by chimeric individuals will be transgenic (Gossler et al., 1986, Robertson, 1987). A transgenic animal has the transgene in all of its cells, although not necessarily expressed. It is not the individual that results originally from the chimeric embryo that is transgenic, but offspring of that individual. This is an important distinction in as much as the chimeric individual can act as founder stock to produce many individuals that carry the desirable gene(s).
Early results have shown that use of chimeras is effective for producing transgenic mice. About 70% of expanded mouse blastocysts develop into live young with about 50% of the young born being chimeric (Bradley et al., 1984). Twenty percent of these chimeric young have germ cell chimerism. Utilizing this method it is not unreasonable to expect that chimerism in the germ line may be 20-30%.
The generation of transgenic animals depends on recombination of the exogenous DNA providing the transgene, with endogenous DNA. Although the majority of such recombinational events are non-homologous reactions, many cell types (including ES-cells) also possesses the enzymatic machinery required for homologous recombination. The homology-dependent recombination between exogenous DNA and chromosomal DNA sequences is referred to as gene targeting, and it offers an additional dimension to transgenic technology. Gene targeting allows the transfer of genetic alterations/mutations created in vitro to precise sites within the host cellular genome. If the host cells are pluripotent ES-cells, such alterations can then be transferred to the germ line of a living organism.
ES cells have been used to produce transgenic lines of mice which through homologous recombination have directed gene insertion. This strategy of creating animals with specific genomic changes has immense potential in agriculture, and in furthering our understanding of the genetic control of mammalian development. However, the ES-cell method has not been successfully applied to production of larger transgenic mammals, for example, transgenic swine. A reason for the failure to extrapolate methods from mice to swine is the difference in developmental stages of the species. For example, the embryonic disc is not a solid mass in swine as in a 5-day old mouse. Other methods include embryonic infection with a recombinant molecule, for example, a retroviral vector with a transgene. Piedrahita et al. (1988, 1990) isolated potential swine stem cells, but was unable to maintain lines or demonstrate these cells' pluripotentiality.
Attempts to use embryonic carcinoma cells to produce chimeric mice, by introducing such cells into an embryo have had limited success. Martin (1981) reported growing mouse stem cells in media conditioned by the growth of teratocarcinoma cells. However, employing cancer cells in a growth environment is not likely to be palatable to the general public if such transgenic animals are to be used for products for human use, for example, food or organs for transplants.
Notarianni et al. (1990) report methods to produce transgenic pigs by use of pluripotent stem cells but do not convincingly show that pluripotent embryonic stem cells were produced. Chimeric pigs were not reported as an intermediate step toward production of a transgenic pig. Pluripotent cells are defined as cells that are capable of being induced to develop into several different cell types. True totipotent embryonic cell types are those capable of being induced to develop into any cell type present in an entire animal.
The morphological description and figures illustrating some of the "selected" cells" in the Evans patent application, International Publication No. WO90/03432, and publications from his group are more reminiscent of epithelial cells, than of embryonic stem cells from other organisms such as the mouse. Indeed, the authors state the "ES" cells from pigs are morphologically dissimilar from mouse ES-cells. Also, no biochemical tests were done to confirm that the selected cells were not differentiated. The only evidence of pluripotency was production of differentiated cells in culture.
Chimeric pigs may be made in several ways, for example, by fusion of aggregates of embryonic cells such as morulas. Transgenic pigs are not derived from chimeras made in this fashion, because the fused cells maintain their genetic complements.
Even if some embryonic stem cells were actually mixed into the "selected" cell population reported by Evans, use of these cell populations to produce chimeric pigs would be expected to be relatively inefficient because chance would dictate whether an embryonic stem cell would be included in the injected material. The probability of inclusion would be expected to be proportional to the percentage of embryonic stem cells in the mixed culture. The lack of a homogeneous culture would lead to inefficient and unpredictable results. Moreover, the method disclosed could not be described as "a method to produce embryonic stem cells," which implies homogeneity and reproducibility.
Evans teaches that a feeder cell layer is necessary for cell growth and teaches away from the use of conditioned medium or growth factors. A simpler culture method is desirable to reduce costs and improve throughput. A feeder layer and the use of conditioned media were also part of the methods of Piedrahita et al. (1988, 1990a and b and Gossler (1986). The Piedrahita et al. 1990a reference teaches away from conditioned medium.
Strojek (1990) describes methods and results similar to those of Evans. Trophoblastic cells and non-homogeneous cultures derived from swine embryos were disclosed.
Handyside (1987) attempted to produce chimeric sheep from embryonic stem cells, but was admittedly unsuccessful. Flake (1986) produced chimeras, but resorted to in utero transplant. Doetschman (1988) identified "embryonic stem cells" from hamsters by growing them on mouse embryonic fibroblast feeder layers. Pluripotency was determined by differentiation in suspension cultures. Ware (1988) reported embryo derived cells from "farm animals" growing on Buffalo Rat Liver BRL and mouse primary fetal fibroblasts.
Wall (1991) suggested using transgenic swine as factories to produce biological products, but did not teach how to accomplish this goal.
Improved methods for the production of transgenic pigs are needed. Transgenic animals are useful as models for diseases for the testing of pharmacological agents prior to clinical trials or the testing of therapeutic modalities. Another advantage is that more desirable qualities in farm animals may be produced by introducing desirable transgenes that can provide such desirable products. These desirable qualities include increased efficiency in feed utilization, improved meat quality, increased pest and disease resistance, and increased fertility.
Transgenic animals are an alternative "factory" for making useful proteins by recombinant genetic techniques. Large animals such as pigs, are potential factories for some products not obtainable from recombinant hosts that are microorganisms or small animals. An example of such products are organs which are transplantable into humans.
Embryonic stem cell transfer to produce transgenic pigs is an improvement over available methods. A reason that embryonic stem cell-mediated gene transfer has not been employed in domestic livestock is the lack of established, stable embryonic stem cell lines available from these species. The availability of such lines would provide feasible methods to produce transgenic animals.
Previous failures to identify and isolate ES cells in swine, may have been due in part to the expectation that such cells would be fast-growing and resemble those of the mouse. In some reports, malignant transformation was necessary to overcome the inherent quiescence of the embryonic disc. McWhir (1989).
Another problem in extrapolating from mice to ungulates, such as swine, is that exactly analogous stages do not exist in the embryos of mice and of ungulates owing to differences in their development. In ungulates, growth is generally slower, and the early embryonic ectoderm is present in a discoid arrangement and not as a solid mass as in the 5-day old mouse embryo.
In the present invention, limitations of the art are overcome by the production of stable, pluripotent swine embryonic stem cell cultures. These cell cultures are used to make chimeric pigs, an intermediate step in producing a transgenic pig. The invention differs in the stage of development of the host into which embryonic cells are introduced, in culture conditions, validation of potency, and production of a chimera.